Bicyclic peptide ligands specific for mt1-mmp

ABSTRACT

The present invention relates to polypeptides which are covalently bound to aromatic molecular scaffolds such that two or more peptide loops are subtended between attachment points to the scaffold. In particular, the invention describes peptides which are high affinity binders of membrane type 1 metalloprotease (MT1-MMP), such as the collagen binding site of MT1-MMP. The invention also describes drug conjugates comprising said peptides, conjugated to one or more effector and/or functional groups which have utility in imaging and targeted cancer therapy.

FIELD OF THE INVENTION

The present invention relates to polypeptides which are covalently boundto aromatic molecular scaffolds such that two or more peptide loops aresubtended between attachment points to the scaffold. In particular, theinvention describes peptides which are high affinity binders of membranetype 1 metalloprotease (MT1-MMP), such as the collagen binding site ofMT1-MMP. The invention also describes drug conjugates comprising saidpeptides, conjugated to one or more effector and/or functional groupswhich have utility in imaging and targeted cancer therapy.

BACKGROUND OF THE INVENTION

Cyclic peptides are able to bind with high affinity and targetspecificity to protein targets and hence are an attractive moleculeclass for the development of therapeutics. In fact, several cyclicpeptides are already successfully used in the clinic, as for example theantibacterial peptide vancomycin, the immunosuppressant drugcyclosporine or the anti-cancer drug octreotide (Driggers et al. (2008),Nat Rev Drug Discov 7 (7), 608-24). Good binding properties result froma relatively large interaction surface formed between the peptide andthe target as well as the reduced conformational flexibility of thecyclic structures. Typically, macrocycles bind to surfaces of severalhundred square angstrom, as for example the cyclic peptide CXCR4antagonist CVX15 (400 Å²; Wu et al. (2007), Science 330, 1066-71), acyclic peptide with the Arg-Gly-Asp motif binding to integrin αVb3 (355Å²) (Xiong et al. (2002), Science 296 (5565), 151-5) or the cyclicpeptide inhibitor upain-1 binding to urokinase-type plasminogenactivator (603 Å²; Zhao et al. (2007), J Struct Biol 160 (1), 1-10).

Due to their cyclic configuration, peptide macrocycles are less flexiblethan linear peptides, leading to a smaller loss of entropy upon bindingto targets and resulting in a higher binding affinity. The reducedflexibility also leads to locking target-specific conformations,increasing binding specificity compared to linear peptides. This effecthas been exemplified by a potent and selective inhibitor of matrixmetalloproteinase 8 (MMP-8) which lost its selectivity over other MMPswhen its ring was opened (Cherney et al. (1998), J Med Chem 41 (11),1749-51). The favorable binding properties achieved throughmacrocyclization are even more pronounced in multicyclic peptides havingmore than one peptide ring as for example in vancomycin, nisin andactinomycin.

Different research teams have previously tethered polypeptides withcysteine residues to a synthetic molecular structure (Kemp and McNamara(1985), J. Org. Chem; Timmerman et al. (2005), ChemBioChem). Meloen andco-workers had used tris(bromomethyl)benzene and related molecules forrapid and quantitative cyclisation of multiple peptide loops ontosynthetic scaffolds for structural mimicry of protein surfaces(Timmerman et al. (2005), ChemBioChem). Methods for the generation ofcandidate drug compounds wherein said compounds are generated by linkingcysteine containing polypeptides to a molecular scaffold as for exampletris(bromomethyl)benzene are disclosed in WO 2004/077062 and WO2006/078161.

Phage display-based combinatorial approaches have been developed togenerate and screen large libraries of bicyclic peptides to targets ofinterest (Heinis et al. (2009), Nat Chem Biol 5 (7), 502-7 and WO2009/098450). Briefly, combinatorial libraries of linear peptidescontaining three cysteine residues and two regions of six random aminoacids (Cys-(Xaa)₆-Cys-(Xaa)₆-Cys) were displayed on phage and cyclisedby covalently linking the cysteine side chains to a small molecule(tris-(bromomethyl)benzene).

SUMMARY OF THE INVENTION

According to a first aspect of the invention, there is provided apeptide ligand specific for the collagen binding site of MT1-MMPcomprising a polypeptide comprising at least three cysteine residues,separated by at least two loop sequences, and an aromatic molecularscaffold which forms covalent bonds with the cysteine residues of thepolypeptide such that at least two polypeptide loops are formed on themolecular scaffold.

According to a further aspect of the invention, there is provided a drugconjugate comprising a peptide ligand as defined herein conjugated toone or more effector and/or functional groups.

According to a further aspect of the invention, there is provided apharmaceutical composition comprising a peptide ligand or a drugconjugate as defined herein in combination with one or morepharmaceutically acceptable excipients.

According to a further aspect of the invention, there is provided apeptide ligand or drug conjugate as defined herein for use inpreventing, suppressing or treating a disease or disorder mediated byMT1-MMP.

DETAILED DESCRIPTION OF THE INVENTION

In one embodiment, said loop sequences comprise 6 amino acids.

In a further embodiment, said loop sequences comprise three cysteineresidues separated by two loop sequences both of which consists of 6amino acids.

In one embodiment, the peptide ligand comprises an amino acid sequenceselected from:

(SEQ ID NO: 1) C_(i)-X₁-X₂-X₃-X₄-X₅-X₆-C_(ii)-L-F-G-X₁₀-Y-X₁₂-C_(iii)

wherein X₁-X₆, X₁₀ and X₁₂ represent any natural or non-natural aminoacid and C_(i), C_(ii) and C_(iii), represent first, second and thirdcysteine residues, respectively or a pharmaceutically acceptable saltthereof.

In one embodiment, X₁ represents S, P, HyP or D.

In one embodiment, X₂ represents F, L, Y, V, H or I.

In one embodiment, X₃ represents D, S or E.

In one embodiment, X₄ represents W, T, R or I.

In one embodiment, X₅ represents W, E, D, S, R, A or H.

In one embodiment, X₆ represents I, T, M, V, L or Q.

In one embodiment, X₁₀ represents D, E, N, S, T or Q.

In one embodiment, X₁₂ represents T, R, S, N, K, D or H.

In one embodiment, the peptide ligand ofC_(i)-X₁-X₂-X₃-X₄-X₅-X₆-C_(ii)-L-F-G-X₁₀-Y-X₁₂-C_(iii) (SEQ ID NO: 1) isselected from:

CPYSWETCLFGDYRC(SEQ ID NO: 2);  C[HyP]YSWETCLFGDYRC(SEQ ID NO: 3);CSLDWETCLFGDYRC(SEQ ID NO: 4);  CDVEWETCLFGDYRC(SEQ ID NO: 5); CPYSWDTCLFGDYRC(SEQ ID NO: 6);  CPHDWETCLFGDYRC(SEQ ID NO: 7); CPYSWDMCLFGDYRC(SEQ ID NO: 8);  CPYSWDVCLFGDYRC(SEQ ID NO: 9); CPYSWDLCLFGDYRC(SEQ ID NO: 10);  CPYSWSQCLFGDYRC(SEQ ID NO: 11); CPYSWSTCLFGDYRC(SEQ ID NO: 12);  CPYSWDICLFGDYRC(SEQ ID NO: 13); CPYSWRTCLFGDYRC(SEQ ID NO: 14):  CPYSWETCLFGDYSC(SEQ ID NO: 15); CPYSWETCLFGEYNC(SEQ ID NO: 16);  CPYSWETCLFGEYKC(SEQ ID NO: 17); CPYSWETCLFGNYTC(SEQ ID NO: 18);  CPYSWETCLFGDYDC(SEQ ID NO: 19); CPYSWETCLFGSYRC(SEQ ID NO: 20);  CPYSWETCLFGSYTC(SEQ ID NO: 21); CPYSWETCLFGTYTC(SEQ ID NO: 22);  CPYDWATCLFGDYRC(SEQ ID NO: 23); CPYDTWTCLFGDYRC(SEQ ID NO: 24);  CPYDRHTCLFGDYRC(SEQ ID NO: 25); CPYDIRTCLFGDYRC(SEQ ID NO: 26);  and CPLSWSTCLFGQYHC(SEQ ID NO: 27).

In a further embodiment, the peptide ligand ofC_(i)-X₁-X₂-X₃-X₄-X₅-X₆-C_(ii)-L-F-G-X₁₀-Y-X₁₂-C_(iii) (SEQ ID NO: 1) isselected from:

-   -   A-(SEQ ID NO: 2)-A (BCY1025);    -   Ac-(SEQ ID NO: 2) (BCY1027);    -   [DOTH]-G-[Sar]₅-(SEQ ID NO: 2) (BCY1388);    -   A-(SEQ ID NO: 3)-A (BCY1029);    -   A-(SEQ ID NO: 4)-A (BCY1030);    -   A-(SEQ ID NO: 5)-A (BCY1031);    -   A-(SEQ ID NO: 6)-A (BCY1032);    -   A-(SEQ ID NO: 7)-A (BCY1034);    -   A-(SEQ ID NO: 8)-A (BCY1035);    -   A-(SEQ ID NO: 9)-A (BCY1036);    -   A-(SEQ ID NO: 10)-A (BCY1037);    -   A-(SEQ ID NO: 11)-A (BCY1038);    -   A-(SEQ ID NO: 12)-A (BCY1039);    -   A-(SEQ ID NO: 13)-A (BCY1040);    -   A-(SEQ ID NO: 14)-A (BCY1041);    -   A-(SEQ ID NO: 15)-A (BCY1042);    -   A-(SEQ ID NO: 16)-A (BCY1043);    -   A-(SEQ ID NO: 17)-A (BCY1044);    -   A-(SEQ ID NO: 18)-A (BCY1045);    -   A-(SEQ ID NO: 19)-A (BCY1046);    -   A-(SEQ ID NO: 20)-A (BCY1047);    -   A-(SEQ ID NO: 21)-A (BCY1048);    -   A-(SEQ ID NO: 22)-A (BCY1049);    -   A-(SEQ ID NO: 23)-A (BCY1051);    -   A-(SEQ ID NO: 24)-A (BCY1052);    -   A-(SEQ ID NO: 25)-A (BCY1053);    -   A-(SEQ ID NO: 26)-A (BCY1054); and    -   A-(SEQ ID NO: 27)-A (BCY1056).

In a further embodiment, the molecular scaffold is TBMB and the peptideligand of C_(i)-X₁-X₂-X₃-X₄-X₅-X₆-C_(ii)-L-F-G-X₁₀-Y-X₁₂-C_(iii) (SEQ IDNO: 1) is selected from:

-   -   A-(SEQ ID NO: 2)-A (BCY1025);    -   Ac-(SEQ ID NO: 2) (BCY1027);    -   [DOTH]-G-[Sar]₅-(SEQ ID NO: 2) (BCY1388);    -   A-(SEQ ID NO: 3)-A (BCY1029);    -   A-(SEQ ID NO: 4)-A (BCY1030);    -   A-(SEQ ID NO: 5)-A (BCY1031);    -   A-(SEQ ID NO: 6)-A (BCY1032);    -   A-(SEQ ID NO: 7)-A (BCY1034);    -   A-(SEQ ID NO: 8)-A (BCY1035);    -   A-(SEQ ID NO: 9)-A (BCY1036);    -   A-(SEQ ID NO: 10)-A (BCY1037);    -   A-(SEQ ID NO: 11)-A (BCY1038);    -   A-(SEQ ID NO: 12)-A (BCY1039);    -   A-(SEQ ID NO: 13)-A (BCY1040);    -   A-(SEQ ID NO: 14)-A (BCY1041);    -   A-(SEQ ID NO: 15)-A (BCY1042);    -   A-(SEQ ID NO: 16)-A (BCY1043);    -   A-(SEQ ID NO: 17)-A (BCY1044);    -   A-(SEQ ID NO: 18)-A (BCY1045);    -   A-(SEQ ID NO: 19)-A (BCY1046);    -   A-(SEQ ID NO: 20)-A (BCY1047);    -   A-(SEQ ID NO: 21)-A (BCY1048);    -   A-(SEQ ID NO: 22)-A (BCY1049);    -   A-(SEQ ID NO: 23)-A (BCY1051);    -   A-(SEQ ID NO: 24)-A (BCY1052);    -   A-(SEQ ID NO: 25)-A (BCY1053);    -   A-(SEQ ID NO: 26)-A (BCY1054); and    -   A-(SEQ ID NO: 27)-A (BCY1056).

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by those of ordinary skillin the art, such as in the arts of peptide chemistry, cell culture andphage display, nucleic acid chemistry and biochemistry. Standardtechniques are used for molecular biology, genetic and biochemicalmethods (see Sambrook et al., Molecular Cloning: A Laboratory Manual,3rd ed., 2001, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y.; Ausubel et al., Short Protocols in Molecular Biology (1999) 4^(th)ed., John Wiley & Sons, Inc.), which are incorporated herein byreference.

Nomenclature

Numbering

When referring to amino acid residue positions within the peptides ofthe invention, cysteine residues (C_(i), C_(ii) and C_(iii)) are omittedfrom the numbering as they are invariant, therefore, the numbering ofamino acid residues within the peptides of the invention is referred toas below:

(SEQ ID NO: 2)C_(i)-P₁-Y₂-S₃-W₄-E₅-T₆-C_(ii)-L₇-F₈-G₉-D₁₀-Y₁₁-R₁₂-C_(iii). 

For the purpose of this description, all bicyclic peptides are assumedto be cyclised with either TBMB (1,3,5-tris(bromomethyl)benzene)yielding a tri-substituted 1,3,5-trismethylbenzene structure.Cyclisation with TBMB occurs on C_(i), C_(ii), and C_(iii).

Molecular Format

N- or C-terminal extensions to the bicycle core sequence are added tothe left or right side of the sequence, separated by a hyphen. Forexample, an N-terminal βAla-Sar10-Ala tail would be denoted as:

(SEQ ID NO: X) βAla-Sar10-A-. 

Inversed Peptide Sequences

In light of the disclosure in Nair et al (2003) J Immunol 170(3),1362-1373, it is envisaged that the peptide sequences disclosed hereinwould also find utility in their retro-inverso form. For example, thesequence is reversed (i.e. N-terminus becomes C-terminus and vice versa)and their stereochemistry is likewise also reversed (i.e. D-amino acidsbecome L-amino acids and vice versa).

Peptide Ligands

A peptide ligand, as referred to herein, refers to a peptide covalentlybound to a molecular scaffold. Typically, such peptides comprise two ormore reactive groups (i.e. cysteine residues) which are capable offorming covalent bonds to the scaffold, and a sequence subtended betweensaid reactive groups which is referred to as the loop sequence, since itforms a loop when the peptide is bound to the scaffold. In the presentcase, the peptides comprise at least three cysteine residues (referredto herein as C_(i), C_(ii) and C_(iii)), and form at least two loops onthe scaffold.

Advantages of the Peptide Ligands

Certain bicyclic peptides of the present invention have a number ofadvantageous properties which enable them to be considered as suitabledrug-like molecules for injection, inhalation, nasal, ocular, oral ortopical administration. Such advantageous properties include:

-   -   Species cross-reactivity. Certain ligands demonstrate        cross-reactivity across PBPs from different bacterial species        and hence are able to treat infections caused by multiple        species of bacteria. Other ligands may be highly specific for        the PBPs of certain bacterial species which may be advantageous        for treating an infection without collateral damage to the        beneficial flora of the patient;    -   Protease stability. Bicyclic peptide ligands should ideally        demonstrate stability to plasma proteases, epithelial        (“membrane-anchored”) proteases, gastric and intestinal        proteases, lung surface proteases, intracellular proteases and        the like. Protease stability should be maintained between        different species such that a bicycle lead candidate can be        developed in animal models as well as administered with        confidence to humans;    -   Desirable solubility profile. This is a function of the        proportion of charged and hydrophilic versus hydrophobic        residues and intra/inter-molecular H-bonding, which is important        for formulation and absorption purposes;    -   An optimal plasma half-life in the circulation. Depending upon        the clinical indication and treatment regimen, it may be        required to develop a bicyclic peptide for short exposure in an        acute illness management setting, or develop a bicyclic peptide        with enhanced retention in the circulation, and is therefore        optimal for the management of more chronic disease states. Other        factors driving the desirable plasma half-life are requirements        of sustained exposure for maximal therapeutic efficiency versus        the accompanying toxicology due to sustained exposure of the        agent; and    -   Selectivity. Certain peptide ligands of the invention        demonstrate selectivity for MT1-MMP, but does not cross-react        with MMP isoforms, such as MMP-1, MMP-2, MMP-15 and MMP-16.

Pharmaceutically Acceptable Salts

It will be appreciated that salt forms are within the scope of thisinvention, and references to peptide ligands include the salt forms ofsaid ligands.

The salts of the present invention can be synthesized from the parentcompound that contains a basic or acidic moiety by conventional chemicalmethods such as methods described in Pharmaceutical Salts: Properties,Selection, and Use, P. Heinrich Stahl (Editor), Camille G. Wermuth(Editor), ISBN: 3-90639-026-8, Hardcover, 388 pages, August 2002.Generally, such salts can be prepared by reacting the free acid or baseforms of these compounds with the appropriate base or acid in water orin an organic solvent, or in a mixture of the two.

Acid addition salts (mono- or di-salts) may be formed with a widevariety of acids, both inorganic and organic. Examples of acid additionsalts include mono- or di-salts formed with an acid selected from thegroup consisting of acetic, 2,2-dichloroacetic, adipic, alginic,ascorbic (e.g. L-ascorbic), L-aspartic, benzenesulfonic, benzoic,4-acetamidobenzoic, butanoic, (+) camphoric, camphor-sulfonic,(+)-(1S)-camphor-10-sulfonic, capric, caproic, caprylic, cinnamic,citric, cyclamic, dodecylsulfuric, ethane-1,2-disulfonic,ethanesulfonic, 2-hydroxyethanesulfonic, formic, fumaric, galactaric,gentisic, glucoheptonic, D-gluconic, glucuronic (e.g. D-glucuronic),glutamic (e.g. L-glutamic), α-oxoglutaric, glycolic, hippuric,hydrohalic acids (e.g. hydrobromic, hydrochloric, hydriodic),isethionic, lactic (e.g. (+)-L-lactic, (±)-DL-lactic), lactobionic,maleic, malic, (−)-L-malic, malonic, (±)-DL-mandelic, methanesulfonic,naphthalene-2-sulfonic, naphthalene-1,5-disulfonic,1-hydroxy-2-naphthoic, nicotinic, nitric, oleic, orotic, oxalic,palmitic, pamoic, phosphoric, propionic, pyruvic, L-pyroglutamic,salicylic, 4-amino-salicylic, sebacic, stearic, succinic, sulfuric,tannic, (+)-L-tartaric, thiocyanic, p-toluenesulfonic, undecylenic andvaleric acids, as well as acylated amino acids and cation exchangeresins.

One particular group of salts consists of salts formed from acetic,hydrochloric, hydriodic, phosphoric, nitric, sulfuric, citric, lactic,succinic, maleic, malic, isethionic, fumaric, benzenesulfonic,toluenesulfonic, sulfuric, methanesulfonic (mesylate), ethanesulfonic,naphthalenesulfonic, valeric, propanoic, butanoic, malonic, glucuronicand lactobionic acids. One particular salt is the hydrochloride salt.Another particular salt is the acetate salt.

If the compound is anionic, or has a functional group which may beanionic (e.g., —COOH may be —COO⁻), then a salt may be formed with anorganic or inorganic base, generating a suitable cation. Examples ofsuitable inorganic cations include, but are not limited to, alkali metalions such as Li⁺, Na⁺ and K⁺, alkaline earth metal cations such as Ca²⁺and Mg²⁺, and other cations such as Al³⁺ or Zn⁺. Examples of suitableorganic cations include, but are not limited to, ammonium ion (i.e.,NH₄+) and substituted ammonium ions (e.g., NH₃R⁺, NH₂R₂ ⁺, NHR₃ ⁺, NR₄⁺). Examples of some suitable substituted ammonium ions are thosederived from: methylamine, ethylamine, diethylamine, propylamine,dicyclohexylamine, triethylamine, butylamine, ethylenediamine,ethanolamine, diethanolamine, piperazine, benzylamine,phenylbenzylamine, choline, meglumine, and tromethamine, as well asamino acids, such as lysine and arginine. An example of a commonquaternary ammonium ion is N(CH₃)₄ ⁺.

Where the peptides of the invention contain an amine function, these mayform quaternary ammonium salts, for example by reaction with analkylating agent according to methods well known to the skilled person.Such quaternary ammonium compounds are within the scope of the peptidesof the invention.

Modified Derivatives

It will be appreciated that modified derivatives of the peptide ligandsas defined herein are within the scope of the present invention.Examples of such suitable modified derivatives include one or moremodifications selected from: N-terminal and/or C-terminal modifications;replacement of one or more amino acid residues with one or morenon-natural amino acid residues (such as replacement of one or morepolar amino acid residues with one or more isosteric or isoelectronicamino acids; replacement of one or more non-polar amino acid residueswith other non-natural isosteric or isoelectronic amino acids); additionof a spacer group; replacement of one or more oxidation sensitive aminoacid residues with one or more oxidation resistant amino acid residues;replacement of one or more amino acid residues with an alanine,replacement of one or more L-amino acid residues with one or moreD-amino acid residues; N-alkylation of one or more amide bonds withinthe bicyclic peptide ligand; replacement of one or more peptide bondswith a surrogate bond; peptide backbone length modification;substitution of the hydrogen on the alpha-carbon of one or more aminoacid residues with another chemical group, modification of amino acidssuch as cysteine, lysine, glutamate/aspartate and tyrosine with suitableamine, thiol, carboxylic acid and phenol-reactive reagents so as tofunctionalise said amino acids, and introduction or replacement of aminoacids that introduce orthogonal reactivities that are suitable forfunctionalisation, for example azide or alkyne-group bearing amino acidsthat allow functionalisation with alkyne or azide-bearing moieties,respectively.

In one embodiment, the modified derivative comprises an N-terminaland/or C-terminal modification. In a further embodiment, wherein themodified derivative comprises an N-terminal modification using suitableamino-reactive chemistry, and/or C-terminal modification using suitablecarboxy-reactive chemistry. In a further embodiment, said N-terminal orC-terminal modification comprises addition of an effector group,including but not limited to a cytotoxic agent, a radiochelator or achromophore.

In a further embodiment, the modified derivative comprises an N-terminalmodification. In a further embodiment, the N-terminal modificationcomprises an N-terminal acetyl group. In this embodiment, the N-terminalcysteine group (the group referred to herein as C_(i)) is capped withacetic anhydride or other appropriate reagents during peptide synthesisleading to a molecule which is N-terminally acetylated. This embodimentprovides the advantage of removing a potential recognition point foraminopeptidases and avoids the potential for degradation of the bicyclicpeptide.

In an alternative embodiment, the N-terminal modification comprises theaddition of a molecular spacer group which facilitates the conjugationof effector groups and retention of potency of the bicyclic peptide toits target.

In a further embodiment, the modified derivative comprises a C-terminalmodification. In a further embodiment, the C-terminal modificationcomprises an amide group. In this embodiment, the C-terminal cysteinegroup (the group referred to herein as C_(ii)) is synthesized as anamide during peptide synthesis leading to a molecule which isC-terminally amidated. This embodiment provides the advantage ofremoving a potential recognition point for carboxypeptidase and reducesthe potential for proteolytic degradation of the bicyclic peptide.

In one embodiment, the modified derivative comprises replacement of oneor more amino acid residues with one or more non-natural amino acidresidues. In this embodiment, non-natural amino acids may be selectedhaving isosteric/isoelectronic side chains which are neither recognisedby degradative proteases nor have any adverse effect upon targetpotency.

Alternatively, non-natural amino acids may be used having constrainedamino acid side chains, such that proteolytic hydrolysis of the nearbypeptide bond is conformationally and sterically impeded. In particular,these concern proline analogues, bulky sidechains, C□-disubstitutedderivatives (for example, aminoisobutyric acid, Aib), and cyclo aminoacids, a simple derivative being amino-cyclopropylcarboxylic acid.

In one embodiment, the modified derivative comprises the addition of aspacer group. In a further embodiment, the modified derivative comprisesthe addition of a spacer group to the N-terminal cysteine (C_(i)) and/orthe C-terminal cysteine (C_(ii)).

In one embodiment, the modified derivative comprises replacement of oneor more oxidation sensitive amino acid residues with one or moreoxidation resistant amino acid residues.

In one embodiment, the modified derivative comprises replacement of oneor more charged amino acid residues with one or more hydrophobic aminoacid residues. In an alternative embodiment, the modified derivativecomprises replacement of one or more hydrophobic amino acid residueswith one or more charged amino acid residues. The correct balance ofcharged versus hydrophobic amino acid residues is an importantcharacteristic of the bicyclic peptide ligands. For example, hydrophobicamino acid residues influence the degree of plasma protein binding andthus the concentration of the free available fraction in plasma, whilecharged amino acid residues (in particular arginine) may influence theinteraction of the peptide with the phospholipid membranes on cellsurfaces. The two in combination may influence half-life, volume ofdistribution and exposure of the peptide drug, and can be tailoredaccording to the clinical endpoint. In addition, the correct combinationand number of charged versus hydrophobic amino acid residues may reduceirritation at the injection site (if the peptide drug has beenadministered subcutaneously).

In one embodiment, the modified derivative comprises replacement of oneor more L-amino acid residues with one or more D-amino acid residues.This embodiment is believed to increase proteolytic stability by sterichindrance and by a propensity of D-amino acids to stabilise β-turnconformations (Tugyi et al (2005) PNAS, 102(2), 413-418).

In one embodiment, the modified derivative comprises removal of anyamino acid residues and substitution with alanines. This embodimentprovides the advantage of removing potential proteolytic attack site(s).

It should be noted that each of the above mentioned modifications serveto deliberately improve the potency or stability of the peptide. Furtherpotency improvements based on modifications may be achieved through thefollowing mechanisms:

-   -   Incorporating hydrophobic moieties that exploit the hydrophobic        effect and lead to lower off rates, such that higher affinities        are achieved;    -   Incorporating charged groups that exploit long-range ionic        interactions, leading to faster on rates and to higher        affinities (see for example Schreiber et al, Rapid,        electrostatically assisted association of proteins (1996),        Nature Struct. Biol. 3, 427-31); and    -   Incorporating additional constraint into the peptide, by for        example constraining side chains of amino acids correctly such        that loss in entropy is minimal upon target binding,        constraining the torsional angles of the backbone such that loss        in entropy is minimal upon target binding and introducing        additional cyclisations in the molecule for identical reasons.

(for reviews see Gentilucci et al, Curr. Pharmaceutical Design, (2010),16, 3185-203, and Nestor et al, Curr. Medicinal Chem (2009), 16,4399-418).

Isotopic Variations

The present invention includes all pharmaceutically acceptable(radio)isotope-labeled peptide ligands of the invention, wherein one ormore atoms are replaced by atoms having the same atomic number, but anatomic mass or mass number different from the atomic mass or mass numberusually found in nature, and peptide ligands of the invention, whereinmetal chelating groups are attached (termed “effector”) that are capableof holding relevant (radio)isotopes, and peptide ligands of theinvention, wherein certain functional groups are covalently replacedwith relevant (radio)isotopes or isotopically labelled functionalgroups.

Examples of isotopes suitable for inclusion in the peptide ligands ofthe invention comprise isotopes of hydrogen, such as ²H (D) and ³H (T),carbon, such as ¹¹C, ¹³C, and ¹⁴C, chlorine, such as ³⁶Cl, fluorine,such as ¹⁸F, iodine, such as ¹²³I, ¹²⁵I and ¹³¹I, nitrogen, such as ¹³Nand ¹⁵N, oxygen, such as ¹⁵O, ¹⁷O and ¹⁸O, phosphorus, such as ³²P,sulfur, such as ³⁵S, copper, such as ⁶⁴Cu, gallium, such as ⁶⁷Ga or⁶⁸Ga, yttrium, such as ⁹⁰Y and lutetium, such as ¹⁷⁷Lu, and Bismuth,such as ²¹³Bi.

Certain isotopically-labelled peptide ligands of the invention, forexample, those incorporating a radioactive isotope, are useful in drugand/or substrate tissue distribution studies. The peptide ligands of theinvention can further have valuable diagnostic properties in that theycan be used for detecting or identifying the formation of a complexbetween a labelled compound and other molecules, peptides, proteins,enzymes or receptors. The detecting or identifying methods can usecompounds that are labelled with labelling agents such as radioisotopes,enzymes, fluorescent substances, luminous substances (for example,luminol, luminol derivatives, luciferin, aequorin and luciferase), etc.The radioactive isotopes tritium, i.e. ³H (T), and carbon-14, i.e. ¹⁴C,are particularly useful for this purpose in view of their ease ofincorporation and ready means of detection.

Substitution with heavier isotopes such as deuterium, i.e. ²H (D), mayafford certain therapeutic advantages resulting from greater metabolicstability, for example, increased in vivo half-life or reduced dosagerequirements, and hence may be preferred in some circumstances.

Substitution with positron emitting isotopes, such as ¹¹C, ¹⁸F, ¹⁵O and¹³N, can be useful in Positron Emission Topography (PET) studies forexamining target occupancy.

Isotopically-labeled compounds of peptide ligands of the invention cangenerally be prepared by conventional techniques known to those skilledin the art or by processes analogous to those described in theaccompanying Examples using an appropriate isotopically-labeled reagentin place of the non-labeled reagent previously employed.

Aromatic Molecular scaffold

References herein to the term “aromatic molecular scaffold” refer to anymolecular scaffold as defined herein which contains an aromaticcarbocyclic or heterocyclic ring system.

It will be appreciated that the aromatic molecular scaffold may comprisean aromatic moiety. Examples of suitable aromatic moieties within thearomatic scaffold include biphenylene, terphenylene, naphthalene oranthracene.

It will also be appreciated that the aromatic molecular scaffold maycomprise a heteroaromatic moiety. Examples of suitable heteroaromaticmoieties within the aromatic scaffold include pyridine, pyrimidine,pyrrole, furan and thiophene.

It will also be appreciated that the aromatic molecular scaffold maycomprise a halomethylarene moiety, such as a bis(bromomethyl)benzene, atris(bromomethyl)benzene, a tetra(bromomethyl)benzene or derivativesthereof.

Non-limiting examples of aromatic molecular scaffolds include: bis-,tris-, or tetra(halomethyl)benzene; bis-, tris-, ortetra(halomethyl)pyridine; bis-, tris-, or tetra(halomethyl)pyridazine;bis-, tris-, or tetra(halomethyl)pyrimidine; bis-, tris-, ortetra(halomethyl)pyrazine; bis-, tris-, ortetra(halomethyl)-1,2,3-triazine; bis-, tris-, ortetra-halomethyl)-1,2,4-triazine; bis-, tris-, ortetra(halomethyl)pyrrole, -furan, -thiophene; bis-, tris-, ortetra(halomethyl)imidazole, -oxazole, -thiazol; bis-, tris-, ortetra(halomethyl)-3H-pyrazole, -isooxazole, -isothiazol; bis-, tris-, ortetra(halomethyl)biphenylene; bis-, tris-, ortetra(halomethyl)terphenylene; 1,8-bis(halomethyl)naphthalene; bis-,tris-, or tetra(halomethyl)anthracene; and bis-, tris-, ortetra(2-halomethylphenyl)methane.

More specific examples of aromatic molecular scaffolds include:1,2-bis(halomethyl)benzene; 3,4-bis(halomethyl)pyridine;3,4-bis(halomethyl)pyridazine; 4,5-bis(halomethyl)pyrimidine;4,5-bis(halomethyl)pyrazine; 4,5-bis(halomethyl)-1,2,3-triazine;5,6-bis(halomethyl)-1,2,4-triazine; 3,4-bis(halomethyl)pyrrole, -furan,-thiophene and other regioisomers; 4,5-bis(halomethyl)imidazole,-oxazole, -thiazol; 4,5-bis(halomethyl)-3H-pyrazole, -isooxazole,-isothiazol; 2,2′-bis(halomethyl)biphenylene;2,2″-bis(halomethyl)terphenylene; 1,8-bis(halomethyl)naphthalene;1,10-bis(halomethyl)anthracene; bis(2-halomethylphenyl)methane;1,2,3-tris(halomethyl)benzene; 2,3,4-tris(halomethyl)pyridine;2,3,4-tris(halomethyl)pyridazine; 3,4,5-tris(halomethyl)pyrimidine;4,5,6-tris(halomethyl)-1,2,3-triazine; 2,3,4-tris(halomethyl)pyrrole,-furan, -thiophene; 2,4,5-bis(halomethyl)imidazole, -oxazole, -thiazol;3,4,5-bis(halomethyl)-1H-pyrazole, -isooxazole, -isothiazol;2,4,2′-tris(halomethyl)biphenylene;2,3′,2″-tris(halomethyl)terphenylene; 1,3,8-tris(halomethyl)naphthalene;1,3,10-tris(halomethyl)anthracene; bis(2-halomethylphenyl)methane;1,2,4,5-tetra(halomethyl)benzene; 1,2,4,5-tetra(halomethyl)pyridine;2,4,5,6-tetra(halomethyl)pyrimidine; 2,3,4,5-tetra(halomethyl)pyrrole,-furan, -thiophene; 2,2′,6,6′-tetra(halomethyl)biphenylene;2,2″,6,6″-tetra(halomethyl) terphenylene;2,3,5,6-tetra(halomethyl)naphthalene and2,3,7,8-tetra(halomethyl)anthracene; andbis(2,4-bis(halomethyl)phenyl)methane.

As noted in the foregoing documents, the molecular scaffold may be asmall molecule, such as a small organic molecule.

In one embodiment the molecular scaffold may be a macromolecule. In oneembodiment the molecular scaffold is a macromolecule composed of aminoacids, nucleotides or carbohydrates.

In one embodiment the molecular scaffold comprises reactive groups thatare capable of reacting with functional group(s) of the polypeptide toform covalent bonds.

The molecular scaffold may comprise chemical groups which form thelinkage with a peptide, such as amines, thiols, alcohols, ketones,aldehydes, nitriles, carboxylic acids, esters, alkenes, alkynes, azides,anhydrides, succinimides, maleimides, alkyl halides and acyl halides.

In one embodiment, the molecular scaffold may comprise or may consist oftris(bromomethyl)benzene, especially 1,3,5-tris(bromomethyl)benzene(‘TBMB’), or a derivative thereof.

In one embodiment, the molecular scaffold is2,4,6-tris(bromomethyl)mesitylene. This molecule is similar to1,3,5-tris(bromomethyl)benzene but contains three additional methylgroups attached to the benzene ring. This has the advantage that theadditional methyl groups may form further contacts with the polypeptideand hence add additional structural constraint.

The molecular scaffold of the invention contains chemical groups thatallow functional groups of the polypeptide of the encoded library of theinvention to form covalent links with the molecular scaffold. Saidchemical groups are selected from a wide range of functionalitiesincluding amines, thiols, alcohols, ketones, aldehydes, nitriles,carboxylic acids, esters, alkenes, alkynes, anhydrides, succinimides,maleimides, azides, alkyl halides and acyl halides.

Scaffold reactive groups that could be used on the molecular scaffold toreact with thiol groups of cysteines are alkyl halides (or also namedhalogenoalkanes or haloalkanes).

Examples include bromomethylbenzene (the scaffold reactive groupexemplified by TBMB) or iodoacetamide. Other scaffold reactive groupsthat are used to selectively couple compounds to cysteines in proteinsare maleimides, αβ unsaturated carbonyl containing compounds andα-halomethylcarbonyl containing compounds. Examples of maleimides whichmay be used as molecular scaffolds in the invention include:tris-(2-maleimidoethyl)amine, tris-(2-maleimidoethyl)benzene,tris-(maleimido)benzene. An example of an α-halomethylcarbonylcontaining compound isN,N′,N″-(benzene-1,3,5-triyl)tris(2-bromoacetamide). Selenocysteine isalso a natural amino acid which has a similar reactivity to cysteine andcan be used for the same reactions. Thus, wherever cysteine ismentioned, it is typically acceptable to substitute selenocysteineunless the context suggests otherwise.

Effector and Functional Groups

According to a further aspect of the invention, there is provided a drugconjugate comprising a peptide ligand as defined herein conjugated toone or more effector and/or functional groups.

Effector and/or functional groups can be attached, for example, to the Nand/or C termini of the polypeptide, to an amino acid within thepolypeptide, or to the molecular scaffold.

Appropriate effector groups include antibodies and parts or fragmentsthereof. For instance, an effector group can include an antibody lightchain constant region (CL), an antibody CH1 heavy chain domain, anantibody CH2 heavy chain domain, an antibody CH3 heavy chain domain, orany combination thereof, in addition to the one or more constant regiondomains. An effector group may also comprise a hinge region of anantibody (such a region normally being found between the CH1 and CH2domains of an IgG molecule).

In a further embodiment of this aspect of the invention, an effectorgroup according to the present invention is an Fc region of an IgGmolecule. Advantageously, a peptide ligand-effector group according tothe present invention comprises or consists of a peptide ligand Fcfusion having a tβ half-life of a day or more, two days or more, 3 daysor more, 4 days or more, 5 days or more, 6 days or more or 7 days ormore. Most advantageously, the peptide ligand according to the presentinvention comprises or consists of a peptide ligand Fc fusion having atβ half-life of a day or more.

Functional groups include, in general, binding groups, drugs, reactivegroups for the attachment of other entities, functional groups which aiduptake of the macrocyclic peptides into cells, and the like.

The ability of peptides to penetrate into cells will allow peptidesagainst intracellular targets to be effective. Targets that can beaccessed by peptides with the ability to penetrate into cells includetranscription factors, intracellular signalling molecules such astyrosine kinases and molecules involved in the apoptotic pathway.Functional groups which enable the penetration of cells include peptidesor chemical groups which have been added either to the peptide or themolecular scaffold. Peptides such as those derived from such as VP22,HIV-Tat, a homeobox protein of Drosophila (Antennapedia), e.g. asdescribed in Chen and Harrison, Biochemical Society Transactions (2007)Volume 35, part 4, p821; Gupta et al. in Advanced Drug Discovery Reviews(2004) Volume 57 9637. Examples of short peptides which have been shownto be efficient at translocation through plasma membranes include the 16amino acid penetratin peptide from Drosophila Antennapedia protein(Derossi et al (1994) J Biol. Chem. Volume 269 p10444), the 18 aminoacid ‘model amphipathic peptide’ (Oehlke et al (1998) Biochim BiophysActs Volume 1414 p127) and arginine rich regions of the HIV TAT protein.Non peptidic approaches include the use of small molecule mimics orSMOCs that can be easily attached to biomolecules (Okuyama et al (2007)Nature Methods Volume 4 p153). Other chemical strategies to addguanidinium groups to molecules also enhance cell penetration(Elson-Scwab et al (2007) J Biol Chem Volume 282 p13585). Smallmolecular weight molecules such as steroids may be added to themolecular scaffold to enhance uptake into cells.

One class of functional groups which may be attached to peptide ligandsincludes antibodies and binding fragments thereof, such as Fab, Fv orsingle domain fragments. In particular, antibodies which bind toproteins capable of increasing the half-life of the peptide ligand invivo may be used.

In one embodiment, a peptide ligand-effector group according to theinvention has a tβ half-life selected from the group consisting of: 12hours or more, 24 hours or more, 2 days or more, 3 days or more, 4 daysor more, 5 days or more, 6 days or more, 7 days or more, 8 days or more,9 days or more, 10 days or more, 11 days or more, 12 days or more, 13days or more, 14 days or more, 15 days or more or 20 days or more.Advantageously a peptide ligand-effector group or composition accordingto the invention will have a tβ half-life in the range 12 to 60 hours.In a further embodiment, it will have a tβ half-life of a day or more.In a further embodiment still, it will be in the range 12 to 26 hours.

In one particular embodiment of the invention, the functional group isselected from a metal chelator, which is suitable for complexing metalradioisotopes of medicinal relevance.

Possible effector groups also include enzymes, for instance such ascarboxypeptidase G2 for use in enzyme/prodrug therapy, where the peptideligand replaces antibodies in ADEPT.

In one particular embodiment of the invention, the functional group isselected from a drug, such as a cytotoxic agent for cancer therapy.Suitable examples include: alkylating agents such as cisplatin andcarboplatin, as well as oxaliplatin, mechlorethamine, cyclophosphamide,chlorambucil, ifosfamide; Anti-metabolites including purine analogsazathioprine and mercaptopurine or pyrimidine analogs; plant alkaloidsand terpenoids including vinca alkaloids such as Vincristine,Vinblastine, Vinorelbine and Vindesine; Podophyllotoxin and itsderivatives etoposide and teniposide; Taxanes, including paclitaxel,originally known as Taxol; topoisomerase inhibitors includingcamptothecins: irinotecan and topotecan, and type II inhibitorsincluding amsacrine, etoposide, etoposide phosphate, and teniposide.Further agents can include antitumour antibiotics which include theimmunosuppressant dactinomycin (which is used in kidneytransplantations), doxorubicin, epirubicin, bleomycin, calicheamycins,and others.

In one further particular embodiment of the invention, the cytotoxicagent is selected from maytansinoids (such as DM1) or monomethylauristatins (such as MMAE).

DM1 is a cytotoxic agent which is a thiol-containing derivative ofmaytansine and has the following structure:

Monomethyl auristatin E (MMAE) is a synthetic antineoplastic agent andhas the following structure:

In one yet further particular embodiment of the invention, the cytotoxicagent is selected from monomethyl auristatin E (MMAE). Data is presentedherein in FIG. 1 and Tables 3 and 4 which demonstrates the effects ofpeptide ligands conjugated to a toxin containing MMAE.

In one embodiment, the cytotoxic agent is linked to the bicyclic peptideby a cleavable bond, such as a disulphide bond or a protease sensitivebond. In a further embodiment, the groups adjacent to the disulphidebond are modified to control the hindrance of the disulphide bond, andby this the rate of cleavage and concomitant release of cytotoxic agent.

Published work established the potential for modifying thesusceptibility of the disulphide bond to reduction by introducing sterichindrance on either side of the disulphide bond (Kellogg et al (2011)Bioconjugate Chemistry, 22, 717). A greater degree of steric hindrancereduces the rate of reduction by intracellular glutathione and alsoextracellular (systemic) reducing agents, consequentially reducing theease by which toxin is released, both inside and outside the cell. Thus,selection of the optimum in disulphide stability in the circulation(which minimises undesirable side effects of the toxin) versus efficientrelease in the intracellular milieu (which maximises the therapeuticeffect) can be achieved by careful selection of the degree of hindranceon either side of the disulphide bond.

The hindrance on either side of the disulphide bond is modulated throughintroducing one or more methyl groups on either the targeting entity(here, the bicyclic peptide) or toxin side of the molecular construct.

In one embodiment, the cytotoxic agent and linker is selected from anycombinations of those described in WO 2016/067035 (the cytotoxic agentsand linkers thereof are herein incorporated by reference).

Synthesis

The peptides of the present invention may be manufactured syntheticallyby standard techniques followed by reaction with a molecular scaffold invitro. When this is performed, standard chemistry may be used. Thisenables the rapid large scale preparation of soluble material forfurther downstream experiments or validation. Such methods could beaccomplished using conventional chemistry such as that disclosed inTimmerman et al (supra).

Thus, the invention also relates to manufacture of polypeptides selectedas set out herein, wherein the manufacture comprises optional furthersteps as explained below. In one embodiment, these steps are carried outon the end product polypeptide made by chemical synthesis.

Peptides can also be extended, to incorporate for example another loopand therefore introduce multiple specificities.

To extend the peptide, it may simply be extended chemically at itsN-terminus or C-terminus or within the loops using orthogonallyprotected lysines (and analogues) using standard solid phase or solutionphase chemistry. Standard (bio)conjugation techniques may be used tointroduce an activated or activatable N- or C-terminus. Alternativelyadditions may be made by fragment condensation or native chemicalligation e.g. as described in (Dawson et al. 1994. Synthesis of Proteinsby Native Chemical Ligation. Science 266:776-779), or by enzymes, forexample using subtiligase as described in (Chang et al. Proc Natl AcadSci USA. 1994 Dec. 20; 91(26):12544-8 or in Hikari et al Bioorganic &Medicinal Chemistry Letters Volume 18, Issue 22, 15 Nov. 2008, Pages6000-6003).

Alternatively, the peptides may be extended or modified by furtherconjugation through disulphide bonds. This has the additional advantageof allowing the first and second peptide to dissociate from each otheronce within the reducing environment of the cell. In this case, themolecular scaffold (e.g. TBMB) could be added during the chemicalsynthesis of the first peptide so as to react with the three cysteinegroups; a further cysteine or thiol could then be appended to the N orC-terminus of the first peptide, so that this cysteine or thiol onlyreacted with a free cysteine or thiol of the second peptide, forming adisulfide-linked bicyclic peptide-peptide conjugate.

Similar techniques apply equally to the synthesis/coupling of twobicyclic and bispecific macrocycles, potentially creating atetraspecific molecule.

Furthermore, addition of other functional groups or effector groups maybe accomplished in the same manner, using appropriate chemistry,coupling at the N- or C-termini or via side chains. In one embodiment,the coupling is conducted in such a manner that it does not block theactivity of either entity.

Pharmaceutical Compositions

According to a further aspect of the invention, there is provided apharmaceutical composition comprising a peptide ligand as defined hereinin combination with one or more pharmaceutically acceptable excipients.

Generally, the present peptide ligands will be utilised in purified formtogether with pharmacologically appropriate excipients or carriers.Typically, these excipients or carriers include aqueous oralcoholic/aqueous solutions, emulsions or suspensions, including salineand/or buffered media. Parenteral vehicles include sodium chloridesolution, Ringer's dextrose, dextrose and sodium chloride and lactatedRinger's. Suitable physiologically-acceptable adjuvants, if necessary tokeep a polypeptide complex in suspension, may be chosen from thickenerssuch as carboxymethylcellulose, polyvinylpyrrolidone, gelatin andalginates.

Intravenous vehicles include fluid and nutrient replenishers andelectrolyte replenishers, such as those based on Ringer's dextrose.Preservatives and other additives, such as antimicrobials, antioxidants,chelating agents and inert gases, may also be present (Mack (1982)Remington's Pharmaceutical Sciences, 16th Edition).

The compounds of the invention can be used alone or in combination withanother agent or agents. The other agent for use in combination may befor example another antibiotic, or an antibiotic ‘adjuvant’ such as anagent for improving permeability into Gram-negative bacteria, aninhibitor of resistance determinants or an inhibitor of virulencemechanisms.

Suitable antibiotics for use in combination with the compounds of theinvention include but are not limited to:

Beta lactams, such as penicillins, cephalosporins, carbapenems ormonobactams. Suitable penicillins include oxacillin, methicillin,ampicillin, cloxacillin, carbenicillin, piperacillin, tricarcillin,flucloxacillin, and nafcillin; suitable cephalosporins includecefazolin, cefalexin, cefalothin, ceftazidime, cefepime, ceftobiprole,ceftaroline, ceftolozane and cefiderocol; suitable carbapenems includemeropenem, doripenem, imipenem, ertapenem, biapenem and tebipenem;suitable monobactams include aztreonam; Lincosamides such as clindamycinand lincomycin;

Macrolides such as azithromycin, clarithromycin, erythromycin,telithromycin and solithromycin;

Tetracyclines such as tigecycline, omadacycline, eravacycline,doxycycline, and minocycline;

Quinolones such as ciprofloxacin, levofloxacin, moxifloxacin, anddelafloxacin;

Rifamycins such as rifampicin, rifabutin, rifalazil, rifapentine, andrifaximin;

Aminoglycosides such as gentamycin, streptomycin, tobramycin, amikacinand plazomicin;

Glycopeptides such as vancomycin, teichoplanin, telavancin, dalbavancin,and oritavancin,

Pleuromutilins such as lefamulin

Oxazolidinones such as linezolid or tedizolid

Polymyxins such as polymyxin B or colistin;

Trimethoprim, iclaprim, sulfamethoxazole;

Metronidazole;

Fidaxomicin:

Mupirocin;

Fusidic acid;

Daptomycin;

Murepavidin;

Fosfomycin; and

Nitrofurantoin.

Suitable antibiotic ‘adjuvants’ include but are not limited to:

agents known to improve uptake into bacteria such as outer membranepermeabilisers or efflux pump inhibitors; outer membrane permeabilisersmay include polymyxin B nonapeptide or other polymyxin analogues, orsodium edetate;

inhibitors of resistance mechanisms such as beta-lactamase inhibitors;suitable beta-lactamase inhibitors include clavulanic acid, tazobactam,sulbactam, avibactam, relebactam and nacubactam; and

inhibitors of virulence mechanisms such as toxins and secretion systems,including antibodies.

The compounds of the invention can also be used in combination withbiological therapies such as nucleic acid based therapies, antibodies,bacteriophage or phage lysins.

The route of administration of pharmaceutical compositions according tothe invention may be any of those commonly known to those of ordinaryskill in the art. For therapy, the peptide ligands of the invention canbe administered to any patient in accordance with standard techniques.Routes of administration include, but are not limited to, oral (e.g., byingestion); buccal; sublingual; transdermal (including, e.g., by apatch, plaster, etc.); transmucosal (including, e.g., by a patch,plaster, etc.); intranasal (e.g., by nasal spray); ocular (e.g., byeyedrops); pulmonary (e.g., by inhalation or insufflation therapy using,e.g., via an aerosol, e.g., through the mouth or nose); rectal (e.g., bysuppository or enema); vaginal (e.g., by pessary); parenteral, forexample, by injection, including subcutaneous, intradermal,intramuscular, intravenous, intraarterial, intracardiac, intrathecal,intraspinal, intracapsular, subcapsular, intraorbital, intraperitoneal,intratracheal, subcuticular, intraarticular, subarachnoid, andintrasternal; by implant of a depot or reservoir, for example,subcutaneously or intramuscularly. Preferably, the pharmaceuticalcompositions according to the invention will be administeredparenterally. The dosage and frequency of administration will depend onthe age, sex and condition of the patient, concurrent administration ofother drugs, counterindications and other parameters to be taken intoaccount by the clinician.

The peptide ligands of this invention can be lyophilised for storage andreconstituted in a suitable carrier prior to use. This technique hasbeen shown to be effective and art-known lyophilisation andreconstitution techniques can be employed. It will be appreciated bythose skilled in the art that lyophilisation and reconstitution can leadto varying degrees of activity loss and that levels may have to beadjusted upward to compensate.

The compositions containing the present peptide ligands or a cocktailthereof can be administered for therapeutic treatments. In certaintherapeutic applications, an adequate amount to accomplish at leastpartial inhibition, suppression, modulation, killing, or some othermeasurable parameter, of a population of selected cells is defined as a“therapeutically-effective dose”. Amounts needed to achieve this dosagewill depend upon the severity of the disease and the general state ofthe patient's own immune system, but generally range from 10 μg to 250mg of selected peptide ligand per kilogram of body weight, with doses ofbetween 100 μg to 25 mg/kg/dose being more commonly used.

A composition containing a peptide ligand according to the presentinvention may be utilised in therapeutic settings to treat a microbialinfection or to provide prophylaxis to a subject at risk of infection egundergoing surgery, chemotherapy, artificial ventilation or othercondition or planned intervention. In addition, the peptide ligandsdescribed herein may be used extracorporeally or in vitro selectively tokill, deplete or otherwise effectively remove a target cell populationfrom a heterogeneous collection of cells. Blood from a mammal may becombined extracorporeally with the selected peptide ligands whereby theundesired cells are killed or otherwise removed from the blood forreturn to the mammal in accordance with standard techniques.

Therapeutic Uses

The bicyclic peptides of the invention have specific utility as highaffinity binders of membrane type 1 metalloprotease (MT1-MMP, also knownas MMP14). More specifically to the collagen binding region of thehemopexin domain (Arkadash et al J. Biol. Chem. 2017, 292(8),3481-3495). MT1-MMP is a transmembrane metalloprotease that plays amajor role in the extracellular matrix remodeling, directly by degradingseveral of its components and indirectly by activating pro-MMP2. MT1-MMPis crucial for tumor angiogenesis (Sounni et al (2002) FASEB J. 16(6),555-564) and is over-expressed on a variety of solid tumours, thereforethe drug conjugates comprising MT1-MMP-binding bicycle peptides of thepresent invention have particular utility in the targeted treatment ofcancer, in particular solid tumours such as non-small cell lungcarcinomas. In one embodiment, the bicyclic peptide of the invention isspecific for human MT1-MMP. In a further embodiment, the bicyclicpeptide of the invention is specific for mouse MT1-MMP. In a yet furtherembodiment, the bicyclic peptide of the invention is specific for humanand mouse MT1-MMP. In a yet further embodiment, the bicyclic peptide ofthe invention is specific for human, mouse and dog MT1-MMP.

Polypeptide ligands of the invention may be employed in in vivotherapeutic and prophylactic applications, in vitro and in vivodiagnostic applications, in vitro assay and reagent applications, andthe like. Ligands having selected levels of specificity are useful inapplications which involve testing in non-human animals, wherecross-reactivity is desirable, or in diagnostic applications, wherecross-reactivity with homologues or paralogues needs to be carefullycontrolled. In some applications, such as vaccine applications, theability to elicit an immune response to predetermined ranges of antigenscan be exploited to tailor a vaccine to specific diseases and pathogens.

Substantially pure peptide ligands of at least 90 to 95% homogeneity arepreferred for administration to a mammal, and 98 to 99% or morehomogeneity is most preferred for pharmaceutical uses, especially whenthe mammal is a human. Once purified, partially or to homogeneity asdesired, the selected polypeptides may be used diagnostically ortherapeutically (including extracorporeally) or in developing andperforming assay procedures, immunofluorescent stainings and the like(Lefkovite and Pernis, (1979 and 1981) Immunological Methods, Volumes Iand II, Academic Press, NY).

The conjugates of the peptide ligands of the present invention willtypically find use in preventing, suppressing or treating cancer, inparticular solid tumours such as non-small cell lung carcinomas.

Thus, according to a further aspect of the invention, there are provideddrug conjugates of the peptide ligand as defined herein for use inpreventing, suppressing or treating cancer, in particular solid tumourssuch as non-small cell lung carcinomas.

According to a further aspect of the invention, there is provided amethod of preventing, suppressing or treating cancer, in particularsolid tumours such as non-small cell lung carcinomas which comprisesadministering to a patient in need thereof a drug conjugate of thepeptide ligand as defined herein.

Examples of cancers (and their benign counterparts) which may be treated(or inhibited) include, but are not limited to tumours of epithelialorigin (adenomas and carcinomas of various types includingadenocarcinomas, squamous carcinomas, transitional cell carcinomas andother carcinomas) such as carcinomas of the bladder and urinary tract,breast, gastrointestinal tract (including the esophagus, stomach(gastric), small intestine, colon, rectum and anus), liver(hepatocellular carcinoma), gall bladder and biliary system, exocrinepancreas, kidney,lung (for example adenocarcinomas, small cell lungcarcinomas, non-small cell lung carcinomas, bronchioalveolar carcinomasand mesotheliomas), head and neck (for example cancers of the tongue,buccal cavity, larynx, pharynx, nasopharynx, tonsil, salivary glands,nasal cavity and paranasal sinuses), ovary, fallopian tubes, peritoneum,vagina, vulva, penis, cervix, myometrium, endometrium, thyroid (forexample thyroid follicular carcinoma), adrenal, prostate, skin andadnexae (for example melanoma, basal cell carcinoma, squamous cellcarcinoma, keratoacanthoma, dysplastic naevus); haematologicalmalignancies (i.e. leukemias, lymphomas) and premalignant haematologicaldisorders and disorders of borderline malignancy includinghaematological malignancies and related conditions of lymphoid lineage(for example acute lymphocytic leukemia [ALL], chronic lymphocyticleukemia [CLL], B-cell lymphomas such as diffuse large B-cell lymphoma[DLBCL], follicular lymphoma, Burkitt's lymphoma, mantle cell lymphoma,T-cell lymphomas and leukaemias, natural killer [NK] cell lymphomas,Hodgkin's lymphomas, hairy cell leukaemia, monoclonal gammopathy ofuncertain significance, plasmacytoma, multiple myeloma, andpost-transplant lymphoproliferative disorders), and haematologicalmalignancies and related conditions of myeloid lineage (for exampleacute myelogenousleukemia [AML], chronic myelogenousleukemia [CML],chronic myelomonocyticleukemia [CMML], hypereosinophilic syndrome,myeloproliferative disorders such as polycythaemia vera, essentialthrombocythaemia and primary myelofibrosis, myeloproliferative syndrome,myelodysplastic syndrome, and promyelocyticleukemia); tumours ofmesenchymal origin, for example sarcomas of soft tissue, bone orcartilage such as osteosarcomas, fibrosarcomas, chondrosarcomas,rhabdomyosarcomas, leiomyosarcomas, liposarcomas, angiosarcomas,Kaposi's sarcoma, Ewing's sarcoma, synovial sarcomas, epithelioidsarcomas, gastrointestinal stromal tumours, benign and malignanthistiocytomas, and dermatofibrosarcomaprotuberans; tumours of thecentral or peripheral nervous system (for example astrocytomas, gliomasand glioblastomas, meningiomas, ependymomas, pineal tumours andschwannomas); endocrine tumours (for example pituitary tumours, adrenaltumours, islet cell tumours, parathyroid tumours, carcinoid tumours andmedullary carcinoma of the thyroid); ocular and adnexal tumours (forexample retinoblastoma); germ cell and trophoblastic tumours (forexample teratomas, seminomas, dysgerminomas, hydatidiform moles andchoriocarcinomas); and paediatric and embryonal tumours (for examplemedulloblastoma, neuroblastoma, Wilms tumour, and primitiveneuroectodermal tumours); or syndromes, congenital or otherwise, whichleave the patient susceptible to malignancy (for example XerodermaPigmentosum).

References herein to the term “prevention” involves administration ofthe protective composition prior to the induction of the disease.“Suppression” refers to administration of the composition after aninductive event, but prior to the clinical appearance of the disease.“Treatment” involves administration of the protective composition afterdisease symptoms become manifest.

Animal model systems which can be used to screen the effectiveness ofthe drug conjugates in protecting against or treating the disease areavailable. The use of animal model systems is facilitated by the presentinvention, which allows the development of polypeptide ligands which cancross react with human and animal targets, to allow the use of animalmodels.

The invention is further described below with reference to the followingexamples.

Examples

Materials and Methods

Peptide Synthesis

Peptide synthesis was based on Fmoc chemistry, using a Symphony peptidesynthesiser manufactured by Peptide Instruments and a Syro IIsynthesiser by MultiSynTech. Standard Fmoc-amino acids were employed(Sigma, Merck), with appropriate side chain protecting groups: whereapplicable standard coupling conditions were used in each case, followedby deprotection using standard methodology.

Alternatively, peptides were purified using HPLC and following isolationthey were modified with 1,3,5-tris(bromomethyl)benzene (TBMB, Sigma).For this, linear peptide was diluted with H₂O up to ˜35 mL, ˜500 μL of100 mM TBMB in acetonitrile was added, and the reaction was initiatedwith ˜5 mL of 1 M NH₄HCO₃ in H₂O. The reaction was allowed to proceedfor ˜30-60 min at RT, and lyophilised once the reaction had completed(judged by MALDI). Once completed, 1 ml of 1M L-cysteine hydrochloridemonohydrate (Sigma) in H₂O was added to the reaction for −60 min at RTto quench any excess TBMB.

Following lyophilisation, the modified peptide was purified as above,while replacing the Luna C8 with a Gemini C18 column (Phenomenex), andchanging the acid to 0.1% trifluoroacetic acid. Pure fractionscontaining the correct TBMB-modified material were pooled, lyophilisedand kept at −20° C. for storage.

All amino acids, unless noted otherwise, were used in theL-configurations.

In some cases peptides are converted to activated disulfides prior tocoupling with the free thiol group of a toxin using the followingmethod; a solution of 4-methyl(succinimidyl 4-(2-pyridylthio)pentanoate)(100 mM) in dry DMSO (1.25 mol equiv) was added to a solution of peptide(20 mM) in dry DMSO (1 mol equiv). The reaction was well mixed and DIPEA(20 mol equiv) was added. The reaction was monitored by LC/MS untilcomplete.

Biological Data

Human Fluorescence Polarisation Competition Binding Assay

Due to its high affinity to the MT1-MMP Hemopexin domain (PEX), thefluoresceinated derivative of 17-88-N006 (SEQ ID NO: 28) can be used forcompetition experiments (using FP for detection). Here, a preformedcomplex of PEX with the fluorescent PEX-binding tracer (in allexperiments—ACPYSWETCLFGDYRCA[Sar]₆[KFI] (17-88-N006) (SEQ ID NO: 28))is titrated with free, non-fluoresceinated bicyclic peptide. Since all17-69-based peptides are expected to bind at the same site, the titrantwill displace the fluorescent tracer from PEX. Dissociation of thecomplex can be measured quantitatively, and the Kd of the competitor(titrant) to the target protein determined. The advantage of thecompetition method is that the affinities of non-fluoresceinatedbicyclic peptides can be determined accurately and rapidly.Concentrations of tracer are usually at the Kd or below (here, 1 nM),and the binding protein (here, hemopexin of MT1-MMP) is at a 15-foldexcess such that >90% of the tracer is bound. Subsequently, thenon-fluorescent competitor bicyclic peptide (usually just the bicyclecore sequence) is titrated, such that it displaces the fluorescenttracer from the target protein. The displacement of the tracer ismeasured and associated with a drop in fluorescence polarisation. Thedrop in fluorescence polarisation is proportional to the fraction oftarget protein bound with the non-fluorescent titrant, and thus is ameasure of the affinity of titrant to target protein.

The raw data is fit to the analytical solution of the cubic equationthat describes the equilibria between fluorescent tracer, titrant, andbinding protein. The fit requires the value of the affinity offluorescent tracer to the target protein, which can be determinedseparately by direct binding FP experiments (see next section). Thecurve fitting was performed using Sigmaplot 12.0 and used as an adaptedversion of the equation described by Zhi-Xin Wang (FEBS Letters (1995)360, 111-114).

Selected peptides of the invention were tested in the above mentionedHuman Fluorescence Polarisation competition binding assay and theresults are shown in Table 1:

TABLE 1 Human MT1-MMP Fluorescence Polarisation Competition BindingBicyclic Peptide Molecular Scaffold K_(i) n BCY1025 TBMB 98.52 ± 29.3410 BCY1027 TBMB 92.67 ± 68.46 3 BCY1029 TBMB 503 1 BCY1030 TBMB 670 1BCY1031 TBMB 1100  1 BCY1032 TBMB 318.5 ± 181.3 2 BCY1034 TBMB 406 1BCY1035 TBMB 391 1 BCY1036 TBMB 103.3 ± 31.52 4 BCY1037 TBMB 908 1BCY1038 TBMB 1500  1 BCY1039 TBMB 177 1 BCY1040 TBMB   88.8 1 BCY1041TBMB 178 1 BCY1042 TBMB 89.45 ± 4.02  2 BCY1043 TBMB 275 1 BCY1044 TBMB109 1 BCY1045 TBMB 155 1 BCY1046 TBMB 510 1 BCY1047 TBMB 223 1 BCY1048TBMB 124 1 BCY1049 TBMB 63.45 ± 8.91  23 BCY1051 TBMB 124.6 ± 63.5  2BCY1052 TBMB 490 1 BCY1053 TBMB 358 1 BCY1054 TBMB 848 1 BCY1056 TBMB287 1 BCY1388 TBMB 81.35 ± 32.63 2

Human Fluorescence Polarisation Direct Binding Assay

Direct Binding Fluorescence Polarisation or Anisotropy Assays areperformed by titrating a constant concentration of fluorescent tracer(here, the fluoresceinated bicyclic peptide to be studied) with itsbinding partner (here, the MT1-MMP hemopexin domain). As theconcentration of binding partner increases during the titration, thepolarisation signal changes in proportion to the fraction of bound andunbound material. This allows determination of dissociation rates (Kd)quantitatively. Assay data can be fit using standard ligand bindingequations.

Typically, concentrations of the tracer are ideally well below the Kd ofthe tracer:titrant pair, and concentrations chosen are usually at ˜1 nMor less. The titrant (binding partner) concentration is varied from 0.1nM up to typically 5 μM. The range is chosen such that the maximumchange in fluorescent polarisation can be observed. Buffers employed arephosphate buffered saline in the presence of 0.01% Tween. Experimentswere run in black 384 well low-bind/low volume plates (Corning 3820),and the fluorescent polarisation signal was measured using a BMGPherastar FS plate reader. Fluorescent tracers referred to in the textare bicyclic peptides that have been fluoresceinated using5,6-carboxyfluorescein. Fluoresceination may be performed on theN-terminal amino group of the peptide, which is separated from thebicycle core sequence by a sarcosine spacer (usually Sar5). This can bedone during Fmoc solid phase synthesis or post-synthetically (aftercyclisation with TBMB and purification) if the N-terminal amino group isunique to the peptide. Fluoresceination can also be performed on theC-terminus, usually on a Lysine introduced as the first C-terminalresidue, which is then separated from the bicycle core sequence by asarcosine spacer (usually Sar6). Thus, N-terminal tracers can have amolecular format described as Fluo-Gly-Sar6-A(BicycleCoreSequence), and(BicycleCoreSequence)-A-Sar6-K(Fluo) for a C-terminally fluoresceinatedconstruct.

Fluorescent tracers used in the Examples include BCY1323-Sar6-K(FI),BCY1326-Sar6-K(FI), BCY3418-Sar6-K(FI), BCY3422-K(FI), andBCY1329-Sar6-K(FI). Due to the acidic nature of the fluorescentpeptides, they were typically prepared as concentrated DMSO stocks, fromwhich dilution were prepared in 100 mM Tris pH 8 buffer.

Selected peptides of the invention were tested in the above mentionedfluorescence polarisation direct binding assay and the results are shownin Table 2:

TABLE 2 Human MT1-MMP Fluorescence Polarisation Direct Binding BicyclicPeptide Molecular Scaffold K_(d) n BCY1323 TBMB 35.86 ± 6.65  20 BCY1325TBMB 140 1 BCY1326 TBMB 218 1 BCY1327 TBMB 98.5 1 BCY1329 TBMB 22.05 ±10.96 6

1. A peptide ligand specific for the collagen binding site of MT1-MMPcomprising a polypeptide comprising at least three cysteine residues,separated by at least two loop sequences, and an aromatic molecularscaffold which forms covalent bonds with the cysteine residues of thepolypeptide such that at least two polypeptide loops are formed on themolecular scaffold.
 2. The peptide ligand as defined in claim 1, whereinsaid loop sequences comprise 6 amino acids.
 3. The peptide ligand asdefined in claim 1 or claim 2, wherein said loop sequences comprisethree cysteine residues separated by two loop sequences both of whichconsists of 6 amino acids.
 4. The peptide ligand as defined in any oneof claims 1 to 3, wherein the peptide ligand comprises an amino acidsequence selected from: (SEQ ID NO: 1)Ci-X₁-X₂-X₃-X₄-X₅-X₆-C_(ii)-L-F-G-X₁₀-Y-X₁₂-C_(iii) 

wherein X₁-X₆, X₁₀ and X₁₂ represent any natural or non-natural aminoacid and C_(i), C_(ii) and C_(iii) represent first, second and thirdcysteine residues, respectively or a pharmaceutically acceptable saltthereof.
 5. The peptide ligand as defined in claim 4, wherein: X₁represents S, P, HyP or D; and/or X₂ represents F, L, Y, V, H or I;and/or X₃ represents D, S or E; and/or X₄ represents W, T, R or I;and/or X₅ represents W, E, D, S, R, A or H; and/or X₆ represents I, T,M, V, L or Q; and/or X₁₀ represents D, E, N, S, T or Q; and/or X₁₂represents T, R, S, N, K, D or H.
 6. The peptide ligand as defined inclaim 4 or claim 5, wherein the peptide ligand ofC_(i)-X₁-X₂-X₃-X₄-X₅-X₆-C_(ii)-L-F-G-X₁₀-Y-X₁₂-C_(iii) (SEQ ID NO: 1) isselected from: CPYSWETCLFGDYRC(SEQ ID NO: 2); C[HyP]YSWETCLFGDYRC(SEQ ID NO: 3);  CSLDWETCLFGDYRC(SEQ ID NO: 4); CDVEWETCLFGDYRC(SEQ ID NO: 5);  CPYSWDTCLFGDYRC(SEQ ID NO: 6); CPHDWETCLFGDYRC(SEQ ID NO: 7);  CPYSWDMCLFGDYRC(SEQ ID NO: 8); CPYSWDVCLFGDYRC(SEQ ID NO: 9);  CPYSWDLCLFGDYRC(SEQ ID NO: 10); CPYSWSQCLFGDYRC(SEQ ID NO: 11);  CPYSWSTCLFGDYRC(SEQ ID NO: 12); CPYSWDICLFGDYRC(SEQ ID NO: 13);  CPYSWRTCLFGDYRC(SEQ ID NO: 14): CPYSWETCLFGDYSC(SEQ ID NO: 15);  CPYSWETCLFGEYNC(SEQ ID NO: 16); CPYSWETCLFGEYKC(SEQ ID NO: 17);  CPYSWETCLFGNYTC(SEQ ID NO: 18); CPYSWETCLFGDYDC(SEQ ID NO: 19);  CPYSWETCLFGSYRC(SEQ ID NO: 20); CPYSWETCLFGSYTC(SEQ ID NO: 21);  CPYSWETCLFGTYTC(SEQ ID NO: 22); CPYDWATCLFGDYRC(SEQ ID NO: 23);  CPYDTWTCLFGDYRC(SEQ ID NO: 24); CPYDRHTCLFGDYRC(SEQ ID NO: 25);  CPYDIRTCLFGDYRC(SEQ ID NO: 26);  and CPLSWSTCLFGQYHC(SEQ ID NO: 27); 

such as: A-(SEQ ID NO: 2)-A (BCY1025); Ac-(SEQ ID NO: 2) (BCY1027);[DOTH]-G-[Sar]₅-(SEQ ID NO: 2) (BCY1388); A-(SEQ ID NO: 3)-A (BCY1029);A-(SEQ ID NO: 4)-A (BCY1030); A-(SEQ ID NO: 5)-A (BCY1031); A-(SEQ IDNO: 6)-A (BCY1032); A-(SEQ ID NO: 7)-A (BCY1034); A-(SEQ ID NO: 8)-A(BCY1035); A-(SEQ ID NO: 9)-A (BCY1036); A-(SEQ ID NO: 10)-A (BCY1037);A-(SEQ ID NO: 11)-A (BCY1038); A-(SEQ ID NO: 12)-A (BCY1039); A-(SEQ IDNO: 13)-A (BCY1040); A-(SEQ ID NO: 14)-A (BCY1041); A-(SEQ ID NO: 15)-A(BCY1042); A-(SEQ ID NO: 16)-A (BCY1043); A-(SEQ ID NO: 17)-A (BCY1044);A-(SEQ ID NO: 18)-A (BCY1045); A-(SEQ ID NO: 19)-A (BCY1046); A-(SEQ IDNO: 20)-A (BCY1047); A-(SEQ ID NO: 21)-A (BCY1048); A-(SEQ ID NO: 22)-A(BCY1049); A-(SEQ ID NO: 23)-A (BCY1051); A-(SEQ ID NO: 24)-A (BCY1052);A-(SEQ ID NO: 25)-A (BCY1053); A-(SEQ ID NO: 26)-A (BCY1054); and A-(SEQID NO: 27)-A (BCY1056).
 7. The peptide ligand as defined in any one ofclaims 1 to 6, wherein the molecular scaffold is TBMB.
 8. The peptideligand as defined in claim 7, wherein the molecular scaffold is TBMB andthe peptide ligand ofC_(i)-X₁-X₂-X₃-X₄-X₅-X₆-C_(ii)-L-F-G-X₁₀-Y-X₁₂-C_(iii) (SEQ ID NO: 1) isselected from: A-(SEQ ID NO: 2)-A (BCY1025); Ac-(SEQ ID NO: 2)(BCY1027); [DOTH]-G-[Sar]₅-(SEQ ID NO: 2) (BCY1388); A-(SEQ ID NO: 3)-A(BCY1029); A-(SEQ ID NO: 4)-A (BCY1030); A-(SEQ ID NO: 5)-A (BCY1031);A-(SEQ ID NO: 6)-A (BCY1032); A-(SEQ ID NO: 7)-A (BCY1034); A-(SEQ IDNO: 8)-A (BCY1035); A-(SEQ ID NO: 9)-A (BCY1036); A-(SEQ ID NO: 10)-A(BCY1037); A-(SEQ ID NO: 11)-A (BCY1038); A-(SEQ ID NO: 12)-A (BCY1039);A-(SEQ ID NO: 13)-A (BCY1040); A-(SEQ ID NO: 14)-A (BCY1041); A-(SEQ IDNO: 15)-A (BCY1042); A-(SEQ ID NO: 16)-A (BCY1043); A-(SEQ ID NO: 17)-A(BCY1044); A-(SEQ ID NO: 18)-A (BCY1045); A-(SEQ ID NO: 19)-A (BCY1046);A-(SEQ ID NO: 20)-A (BCY1047); A-(SEQ ID NO: 21)-A (BCY1048); A-(SEQ IDNO: 22)-A (BCY1049); A-(SEQ ID NO: 23)-A (BCY1051); A-(SEQ ID NO: 24)-A(BCY1052); A-(SEQ ID NO: 25)-A (BCY1053); A-(SEQ ID NO: 26)-A (BCY1054);and A-(SEQ ID NO: 27)-A (BCY1056).
 9. The peptide ligand as defined inany one of claims 1 to 8, wherein the pharmaceutically acceptable saltis selected from the free acid or the sodium, potassium, calcium,ammonium salt.
 10. The peptide ligand as defined in any one of claims 1to 9, wherein the MT1-MMP is human MT1-MMP.
 11. A drug conjugatecomprising a peptide ligand as defined in any one of claims 1 to 10,conjugated to one or more effector and/or functional groups.
 12. Thedrug conjugate as defined in claim 11, conjugated to one or morecytotoxic agents.
 13. The drug conjugate as defined in claim 12, whereinsaid cytotoxic agent is selected from MMAE or DM1.
 14. A pharmaceuticalcomposition which comprises the peptide ligand of any one of claims 1 to10 or the drug conjugate of any one of claims 11 to 13, in combinationwith one or more pharmaceutically acceptable excipients.
 15. Thepharmaceutical composition as defined in claim 14, which additionallycomprises one or more therapeutic agents.
 16. The drug conjugate asdefined in any one of claims 11 to 13, for use in preventing,suppressing or treating a disease or disorder mediated by MT1-MMP.